Cancer treatment using compounds that selectively target polyploid cancer cells for disruption

ABSTRACT

The disclosure provides a compound of Formula I:or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are as described herein. The disclosure also provides methods for identifying a compound that selectively kills polyploid cells, and methods for killing polyploid tumor cells by administering a compound of Formula Ito a patient in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application No. 15/985,115,filed on May 21, 2018, which claims the priority benefit under 35 U.S.C§ 119 to U.S. Provisional Patent Application Ser. No. 62/513,958, titled“CANCER TREATMENT USING COMPOUNDS THAT SELECTIVELY TARGET POLYPLOIDCANCER CELLS FOR DISRUPTION,” filed on Jun. 1, 2017, the disclosure ofwhich is hereby incorporated by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The present disclosure generally relates to cancer treatments and moreparticularly, to compounds and compositions that selectively targetpolyploid cancer cells for disruption.

BACKGROUND OF THE INVENTION

Tumor evolution, underpinned by the enormous intratumor heterogeneity,represents a formidable obstacle that currently prevents the developmentof truly curative treatments for cancer. Significantly elevated genomiccontent in polyploid tumor cells has been proposed to facilitate rapidtumor evolution and the acquisition of therapy resistance in variousincurable cancers. Polyploidy is also a hallmark of radiation inducedmitotic catastrophe and is a common phenomenon occurring in tumor cellswith impaired p53 function following exposure to various cytotoxic,genotoxic and antimitotic agents. There is also an increasingappreciation that cancer cells undergo endoreplication to give rise topolyploidy as a means of survival during mitotic catastrophe orgenotoxic stress.

Although polyploidy was previously thought to be incompatible withcellular proliferation, recent data indicate that at low frequency someof these polyploid cancer cells can actually re-enter into mitotic cellcycles via a process of genome reduction called depolyploidization.Furthermore, both preexisting polyploid cells and treatment-inducedpolyploid cells are capable of tumor formation. These findings suggestthat survival from anticancer treatment by endoreplication andsubsequent depolyploidization provides a mechanism by which cancer cellsbecome resistance to anti-cancer drugs. It has also been postulated thatthis mechanism might contribute to the recurrence of more aggressivecancers, because endoreplication may lead to additional oncogenicalterations resulting from repeated rounds of replication in a cell thatmight have compromised the fidelity of DNA synthesis. Therefore, it ishighly desirable to find a way to kill these polyploid tumor cells.Unfortunately, there is currently no effective therapeutic treatmentthat selectively targets polyploid cells for destruction.

SUMMARY OF THE INVENTION

The disclosure addresses these needs and more through a cell-basedscreening system that exploits Bcl-2 to protect polyploid cells fromboth apoptosis and autophagic cell death when MYC-VX680 syntheticlethality is elicited. This innovative system allows for the conversionof a diploid cell line into a homogeneous population of large polyploidcells for the propose of drug screening. After screening a library ofnatural product extracts in the cell-based system, the natural productembelin was identified from the fruits of Embelia ribes Burm as aselective killer of polyploid cells. This embelin activity has not beenpreviously reported in the literature. The therapeutic efficacy ofembelin was further demonstrated against tumorigenesis of polyploidcells but not pseudo-diploid cells in vitro and in vivo. The datasuggest that embelin and its derivatives, are useful to eliminatepolyploid tumor cells and consequently, prevent tumor relapse through arationale combination with polyploid inducing treatments.

Thus, in one embodiment the disclosure provides a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is independently selected from hydrogen, hydroxyl, amino, thiol,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl, alkyloxy,alkyloxyalkyl, alkylamine, dialkylamine, arylamine, heterocyclyl,alkylheterocyclyl, aryl, alkylaryl, and heteroaryl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy esteryl, and aryl;

R² is independently selected from alkyl, alkenyl, alkynyl, cycloalkyl,alkylcycloalkyl, alkoxy, and alkyloxyalkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy, esteryl, and aryl;

R³ and R⁴ are each independently selected from hydrogen, hydroxyl,amino, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl,alkyloxy, alkyloxyalkyl, alkylamine, dialkylamine, arylamine,heterocyclyl, heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, andheteroaryl, each optionally independently substituted with 1 to 6substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl,or aryl, or

R³ and R⁴, together form a 5 or 6-membered heteroaryl ring or a6-membered aryl ring, each optionally independently substituted with 1to 6 substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl,or aryl.

In another embodiment, the disclosure provides a method of identifying acompound that selectively kills polyploid cells, by converting a diploidcell line into a homogeneous population of polyploid cells; screeningone or more compounds against the homogeneous population of polyploidcells; and identifying a compound that selectively kills the homogeneouspopulation of polyploid cells.

In another embodiment, the disclosure provides a method of killingpolyploid tumor cells, by administering to a patient in need thereof, acompound of Formula I or a pharmaceutically acceptable salt thereof,wherein the compound of Formula I and R¹, R², R³, and R⁴ are asdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure, in accordance with one or more variousembodiments, is described in detail with reference to the followingfigures. The drawings are provided for purposes of illustration only andmerely depict exemplary embodiments of the disclosure. These drawingsare provided to facilitate the reader's understanding of the disclosureand should not be considered limiting of the breadth, scope, size, orapplicability of the disclosure. It should be noted that for clarity andease of illustration these drawings are not necessarily made to scale.

FIG. 1A is a graph which illustrates p53 function in RPE-Myc cells;

FIG. 1B is a graph which illustrates p53 function in RPE-Myc cells;

FIG. 1C is a graph which illustrates RPE cells expressing Myc and activeAkt;

FIG. 2A is a graph which illustrates anti-apoptotic members of the Bcl-2family confer resistance to MYC-VX680 lethality independent of itsanti-apoptotic activity;

FIG. 2B are images of cells which illustrates anti-apoptotic members ofthe Bcl-2 family enhancing survival of polyploid RPEMYC cells elicitedby VX680;

FIG. 2C are western blots which illustrates overexpression of Bcl-2,Bcl-2 G145A and Bcl-xLmut1 in RPE-MYC cells;

FIG. 3A is a graph which illustrates the effect of various smallmolecule drugs (Bcl2 inhibitors) on the viability of polyploid cells;

FIG. 3B is a graph which illustrates Bcl2 inhibitors selectively killspolyploid cells as opposed to diploid cells;

FIG. 4A is a drawing which illustrates the chemical structure ofembelin;

FIG. 4B is a graph which illustrates embelin elictingpolyploidy-selective lethal activity as opposed to diploid cells;

FIG. 5A is a graph which illustrates suppression of tumorigenesis ofpolyploid RPEMYC/Bcl2 cells by embelin;

FIG. 5B is a graph which illustrates suppression of tumorigenesis ofpolyploid breast cancer cells by embelin;

FIG. 5C is a graph which illustrates embelin selectively killingpolyploid breast cancer cells as opposed to diploid cells;

FIG. 6A is a graph which illustrates Aurora kinase inhibitor MLN8237elicits cytostatic effect on lung cancer cells;

FIG. 6B shows images of cells which illustrates MM-BRAF cells developpolyploidy when exposed to MLN8237;

FIG. 6C shows images of tumors from selected tissues derived frompolyploid MM-BRAF cells which illustrates version to small, diploidtumor cells;

FIG. 6D is a diagram which illustrates the experimental design whereMM-BRAF cells were induced with MLN8237 in vitro for polyploid cells,followed by subcutaneous implantation into nude mice;

FIG. 6E is a graph which illustrates the percentage of mice with tumorsafter implantation of polyploid MM-BRAF cells;

FIG. 6F is a graph which illustrates the number of mice with metastatictumors in the indicated tissues after implantation of polyploid MM-BRAFcells;

FIG. 7A is a graph which illustrates embelin suppressing tumorigenesisand metastasis of polyploid MM-BRAF cells induced by MLN8237;

FIG. 7B is a graph which illustrates embelin failing to suppresstumorigenesis and metastasis of MM-BRAF cells; and

FIG. 7C is a graph which illustrates embelin selectively killingpolyploid lung cancer cells as opposed to diploid cells.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following description is presented to enable a person of ordinaryskill in the art to make and use embodiments described herein.Descriptions of specific devices, techniques, and applications areprovided only as examples. Various modifications to the examplesdescribed herein will be readily apparent to those of ordinary skill inthe art, and the general principles defined herein may be applied toother examples and applications without departing from the spirit andscope of the disclosure. The word “exemplary” is used herein to mean“serving as an example illustration.” Any aspect or design describedherein as “exemplary” is not necessarily to be construed as preferred oradvantageous over other aspects or designs. Thus, the present disclosureis not intended to be limited to the examples described herein and shownbut is to be accorded the scope consistent with the claims.

As used herein, reference to any biological drug includes any fragment,modification or variant of the biologic, including any pegylated form,glycosylated form, lipidated form, cyclized form or conjugated form ofthe biologic or such fragment, modification or variant or prodrug of anyof the foregoing. As used herein, reference to any small molecule drugincludes any salt, acid, base, hydrate, solvate, ester, isomer, orpolymorph thereof or metabolite or prodrug of any of the foregoing.

It should be understood that the specific order or hierarchy of steps inthe process disclosed herein is an example of exemplary approaches.Based upon design preferences, it is understood that the specific orderor hierarchy of steps in the processes may be rearranged while remainingwithin the scope of the present disclosure. Any accompanying methodclaims present elements of the various steps in a sample order and arenot meant to be limited to the specific order or hierarchy presented.

Abbreviations used herein have their conventional meaning within thechemical and biological arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e. unbranched) or branched chain,or cyclic hydrocarbon radical, or combination thereof, which may befully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated (i.e.C₁-C₁₀ means one to ten carbons). Examples of saturated hydrocarbonradicals include, but are not limited to, groups such as methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds.

Examples of unsaturated alkyl groups include, but are not limited to,vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. Alkyl groups which arelimited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkyl, as exemplified, but not limited,by —CH₂CH₂CH₂CH₂—, —CH₂CH═CHCH₂—, —CH₂CsCCH₂—, —CH₂CH₂CH(CH₂CH₂CH₃)CH₂—.Typically, an alkyl (or alkylene) group will have from 1 to 24 carbonatoms, with those groups having 10 or fewer carbon atoms being preferredin the present invention. A “lower alkyl” or “lower alkylene” is ashorter chain alkyl or alkylene group, generally having eight or fewercarbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S, and wherein the nitrogen,phosphorus, and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N, P andS and Si may be placed at any interior position of the heteroalkyl groupor at the position at which alkyl group is attached to the remainder ofthe molecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CHs)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂₉—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —OCH₃, —OCH₂CH₃, and —CN. Up to twoor three heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term “heteroalkylene”by itself or as part of another substituent means a divalent radicalderived from heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, andthe like). Still. further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)OR¹— represents both —C(O)OR′— and —R¹OC(O). As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR¹, —NR′R″, —OR′, —SR^(′), and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R^(″) or the like, it will be understoodthat the terms heteroalkyl and —NR¹R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyi” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyi include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene” and“heterocycloalkylene” refer to the divalent derivatives of cycloalkyiand heterocycloalkyl, respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (preferably from 1 to 3 rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms (in each separate ring in the caseof multiple rings) selected from N, O, and S, wherein the nitrogen andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a carbon or heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3- isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyI,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryland heteroaryl ring systems are selected from the group of acceptablesubstituents described below. The terms “arylene” and “heteroarylene”refer to the divalent radicals of aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxo, arylthioxo, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, naphthyloxy)propyl, and the like). However, the term“haloaryl,” as used herein is meant to cover only aryls substituted withone or more halogens.

Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specificnumber of members (e.g. “3 to 7 membered”), the term “member” referrersto a carbon or heteroatom.

The term “oxo” as used herein means an oxygen that is double bonded to acarbon atom.

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and“heterocycloalkyl”. “aryl,” “heteroaryl” as well as their divalentradical derivatives) are meant to include both substituted andunsubstituted forms of the indicated radical. Preferred substituents foreach type of radical are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative radicals (including those groupsoften referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR¹, ═O, ═NR′, ═N—OR′, —NR¹R″, —SR′,-halogen, —SiR¹R¹¹R″¹, —OC(O)R¹, —C(O)R″, —CO₂RVC(O)NR¹R″, —OC(O)NR¹R″,—NR¹¹C(O)R¹, —NR′—C(O)NR″R^(m), —NR¹¹C(O)OR¹, —NR—C(NR¹R¹O═NR¹″,—S(O)R′, —S(O)₂R′, —S(O)₂NR¹R″, —NRSO₂R¹, —CN and —NO₂ in a numberranging from zero to (2m^(f)+1), where m¹ is the total number of carbonatoms in such radical. R′, R″, R¹′¹ and R″″ each preferablyindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R¹, R″, R¹¹¹ and R″″ groupswhen more than one of these groups is present. When R¹ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a A-, 5-, 6-, or 7-membered ring. For example,—NR¹R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for alkyl radicals above,exemplary substituents for aryl and heteroaryl groups (as well as theirdivalent derivatives) are varied and are selected from, for example:halogen, —OR¹, —NR¹R″, —SR′, -halogen, —SiR¹R¹¹R¹¹′, —OC(O)R′, —C(O)R¹,—CO₂R′, —C(O)NR¹R″, —OC(O)NR¹R″, —NR¹¹C(O)R¹, —NR{circumflex over( )}C(O)NR¹¹R″′, —NR¹¹C(O)OR′, —NR—C(NR¹R¹¹R¹¹O═NR″¹¹,—NR—C(NR¹R¹O═NR¹″, —S(O)R″, —S(O)₂R′, —S(O)₂NR¹R″, —NRSO₂R¹, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxo, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onaromatic ring system; and where R′, R″, R″′ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl. When a compound of the invention includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R″′ and R″″ groups when more than one of these groupsis present.

Two of the substituents on adjacent atoms of aryl or heteroaryl ring mayoptionally form a ring of the formula —T—C(O)—(CRR′)_(q)—U—, wherein Tand U are independently —NR—, —O—, —CRR¹— or a single bond, and q is aninteger of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂VB—, wherein A and B areindependently —CRR¹—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR¹)_(s)X¹—(C″R″′)d-₃ where s and d are independentlyintegers of from 0 to 3, and X¹ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—,Or-S(O)₂NR¹—. The substituents R, R¹, R″ and R″′ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl.

As used herein, the term “heteroatom” or “ring heteroatom” is meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

An “aminoalkyl” as used herein refers to an amino group covalently boundto an alkylene linker. The amino group is —NR¹R″, wherein R¹ and R″ aretypically selected from hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

(A) —OH₅—NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl, substituted with at least one substituent selected from:

(i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroary], and

(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl, substituted with at least one substituent selected from:

(a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, orheteroaryl, substituted with at least one substituent selected from oxo,—OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

A “size-limited substituent” or “ size-limited substituent group,” asused herein means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted Cj-C_(2O) alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

A “lower substituent” or “ lower substituent group,” as used hereinmeans a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-Cg alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted Cs-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

The compounds of the present invention may exist as salts. The presentinvention includes such salts. Examples of applicable salt forms includehydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates,(−)-tartrates or mixtures thereof including racemic mixtures,succinates, benzoates and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in art.Also included are base addition salts such as sodium, potassium,calcium, ammonium, organic amino, or magnesium salt, or a similar salt.When compounds of the present invention contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples of acceptableacid addition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived organic acids like acetic, propionic,isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric,lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like. Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are encompassedwithin the scope of the present invention.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituent moieties found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike {see, for example, Berge et ah, “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to. provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

The terms “a,” “an,” or “a(n)”_(}) when used in reference to a group ofsubstituents herein, mean at least one. For example, where a compound issubstituted with “an” alkyl or aryl, the compound is optionallysubstituted with at least one alkyl and/or at least one aryl. Moreover,where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different.

The description of the compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocyclo alkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

The terms “treating” or “treatment” in reference to a particular diseaseincludes prevention of the disease.

The symbol — ˜ denotes the point of attachment of a moiety to theremainder of the molecule.

As used herein, the phrase “polyploidy inducing agent’ refers to atherapeutic compound of any type (e.g., nonselective or selective),including small molecules, antibodies, antisense oligonucleotides, smallinterfering RNAs, microRNA-based compounds that induce polyploidy in oneor more cells. Methods for determining the induction or evidence ofpolyploidy in one or more cells can be obtained using routine techniquesknown in the art. For example, evidence of polyploidy can be determinedby detecting elevated expression of p53. p53 is a surrogate forpolyploidization in cells harboring wildtype p53 (See, Gizatullin, F. etal., “The Aurora Kinase inhibitor VX-680 induces endoreduplication andapoptosis preferentially in cells with compromised p53-dependentpostmitotic checkpoint function.” Cancer Res. 66, 7668-77. (2006)).Additionally, cells in which polyploidy has been induced exhibit a grossmorphological increase in cell size and multinucleation, both of whichcan be detected using routine techniques known in the art.

Examples of polyploidy inducing agents, include, but are not limited to,Aurora Kinase inhibitors, microtubule inhibitors (such as, for example,Taxotere, Vincristine, nocodazole, paclitaxel or colcemid), pan-kinaseinhibitors (such as, for example, staurosporine), oncolytic viruses(such as, for example, ONYX-015), Acridine orange, Dolastain-10,Noscapine, topoisomerase II inhibitors (such as, for example, ICRF-187or ICRF-193), 2-4-(7-chloro-2-quinoxalinyl) oxyphenoxypropionic acid,2-4-(7-bromo-2-quinolinyl)oxyphenoxypropionic acid, Platycodin D,microtubule poisons (such as, for example, JG-03-14), actinpolymerization inhibitors (such as, for example, Cytochalasin B),Bistramide A or antitumor antibiotics (Such as, for example, MithramycinSKI).

As used herein, an “Aurora Kinase inhibitor” refers to a therapeuticcompound of any type (e.g., non-selective or selective), including smallmolecules, antibodies, antisense oligos, small interfering RNA, ormicroRNA-based compounds, that suppresses the activity of at least onemember of the Aurora Kinase family, which includes Aurora A, Aurora B,and Aurora C.

The methods of the present disclosure are useful with any known orhereafter developed Aurora Kinase A inhibitor. Examples of an AuroraKinase A inhibitor are PHA-73.9358, MLN-8054, R-763, JNJ-7706621, MP-529and MP-235.

The methods of the present disclosure are useful with any known orhereafter developed Aurora Kinase B inhibitor. Examples of an AuroraKinase B inhibitor are AZD1152, ZM447439, VX-680/MK0457 and Hesperadin.AZD1152, also known as,2-3-(4-(5-2-(3-Fluorophenyl)amino-2-oxoethyl)-1H-pyrazol-3-yl)aminoquinazolin-7yl)oxy)propyl)(ethyl)-aminoethyl dihydrogen phosphate, is a prodrug of a pyrazoloquinazolineAurora Kinase inhibitor (AZD1152-hydroxyquinazoline pyrazolanilide(HQPA)) and is converted rapidly to the active AZD1152-HQPA in plasma(See, Mortlock, A A. et al., J. Med. Chem., 50:2213-24 (2007)).AZD1152-HQPA is a highly potent and selective inhibitor of Aurora B.ZM447439, also known as 4-(4-(N-benzoylamino)anilino)-6-methoxy-7-(3-(1-morpholino)propoxy)guinaZoline, is aquinazoline derivative, inhibits Aurora A and Aurora B. VX-680/MK0457 isa cyclopropane carboxylic acid of{4-4-(4-methyl-piperazin-1-yl)-6-(5-methyl-2H-pyrazol-3-ylamino)-pyrimidin-2-ylsulphanyl-phenyl-amide and inhibits Aurora A, Aurora Band Aurora C. Hesperadin, an indolinone, inhibits Aurora B.

The methods of the present disclosure are useful with any known orhereafter developed Aurora Kinase C inhibitor. Examples of an AuroraKinase C inhibitor are AZD1152 and VX-680/MK-0457.

Frequently induced by a variety of anticancer treatments, polyploidy isknown to confer tumor cells with multi-drug resistance and is believedto be responsible for tumor relapse. Currently, there are not anyclinically approved anticancer drugs that can target polyploid tumorcells for elimination.

To address these needs, the disclosure provides a cell-based assay toscreen for small molecules that can selectively kill polyploid tumorcells, as opposed to their parental pseudo-diploid cells. Applying anextract library of natural products to the screening assay, thehydroxybenzoquinone known as embelin, isolated from the fruits ofEmbelia ribes Burm, was identified as a selective agent againstpolyploid tumor cells. In three different tumor types tested, embelintriggered the rapid demise of polyploid cancer cells but not theirparental pseudo-diploid cells in each cell line in vitro. In all threecases, embelin also suppressed tumorigenesis of polyploid cancer cellsbut not their parental cells in vivo. Collectively, this data reveals apreviously unknown lethal activity of embelin selectively againstpolyploid cells, pioneering the concept that the combination ofpolyploid-inducing treatment with embelin or one of its derivatives,could be a viable strategy for the treatment of a variety of humancancers, and provide the first example of a therapeutic drug combinationthat specifically exploits polyploidization.

Accordingly, in one embodiment the disclosure provides a compound ofFormula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is independently selected from hydrogen, hydroxyl, amino, thiol,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl, alkyloxy,alkyloxyalkyl, alkylamine, dialkylamine, arylamine, heterocyclyl,alkylheterocyclyl, aryl, alkylaryl, and heteroaryl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy esteryl, and aryl;

R² is independently selected from alkyl, alkenyl, alkynyl, cycloalkyl,alkylcycloalkyl, alkoxy, and alkyloxyalkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy, esteryl, and aryl;

R³ and R⁴ are each independently selected from hydrogen, hydroxyl,amino, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl,alkyloxy, alkyloxyalkyl, alkylamine, dialkylamine, arylamine,heterocyclyl, heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, andheteroaryl, each optionally independently substituted with 1 to 6substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl,or aryl, or

R³ and R⁴ together form a 5 or 6-membered heteroaryl ring or a6-membered aryl ring, each optionally independently substituted with 1to 6 substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl,or aryl.

In another embodiment, the disclosure provides a compound of Formula I,wherein:

R¹ is independently selected from hydrogen, hydroxyl, amino, thiol,(C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl,(C₅-C₂₀)cycloalkenyl, (C₈-C₂₀)cycloalkynyl,(C₁-C₂₀)alkyl(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkyl(C₅-C₂₀)cyclo-alkenyl,(C₁-C₂₀)alkyl(C₈-C₂₀)cycloalkynyl, (C₁-C₂₀)alkyloxy,(C₁-C₂₀)alkyloxy(C₁-C₂₀)alkyl, (C₁-C₂₀)alkylamine, (C₁-C₂₀)dialkylamine,arylamine, heterocyclyl, (C₁-C₂₀)alkylhetero-cyclyl, aryl,(C₁-C₂₀)alkylaryl, and heteroaryl, each optionally independentlysubstituted with 1 to 6 substituents selected from hydrogen, halogen,nitro, amino, cyano, isocyano, thiol, hydroxyl, (C₁-C₆)alkyl,(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, aryloxy, esteryl, and aryl;

R² is independently selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkyl(C₃-C₂₀)cycloalkyl,(C₁-C₂₀)alkoxy, and (C₁-C₂₀)alkyloxy(C₁-C₂₀)alkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,(C₁-C₂₀)alkyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, aryloxy, esteryl, andaryl;

R³ and R⁴ are each independently selected from hydrogen, hydroxyl,amino, thiol, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl,(C₃-C₂₀)cycloalkyl, (C₅-C₂₀)cycloalkenyl, (C₈-C₂₀)cycloalkynyl,(C₁-C₂₀)alkyl(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkyl(C₅-C₂₀)cycloalkenyl,(C₁-C₂₀)alkyl(C₈-C₂₀)cycloalkynyl, (C₁-C₂₀)alkyloxy,(C₁-C₂₀)alkyloxy(C₁-C₂₀)alkyl, (C₁-C₂₀)alkylamine, (C₁-C₂₀)dialkylamine,arylamine, heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, andheteroaryl, each optionally independently substituted with 1 to 6substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, (C₁-C₂₀)alkyl, (C₃-C₂₀)cycloalkyl,(C₁-C₂₀)alkoxy, aryloxy, esteryl, and aryl, or

R³ and R⁴ together form a 5 or 6-membered heterocyclic or heteroarylring or a 6-membered aryl ring, each optionally independentlysubstituted with 1 to 6 substituents selected from hydrogen, halogen,nitro, amino, cyano, isocyano, thiol, hydroxyl, (C₁-C₂₀)alkyl,(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, aryloxy, esteryl, and aryl.

In another embodiment, the disclosure provides a compound of Formula I,wherein:

R³ and R⁴ together form a 5-membered heterocyclic ring selected fromsubstituted or unsubstituted pyrrolidine, substituted or unsubstitutedpyrazolidine, substituted or unsubstituted imidazolidine, substituted orunsubstituted tetrahydrofuran, and substituted or unsubstitutedtetrahydrothiophene, or

R³ and R⁴ together form a 5-membered heteroaryl ring selected fromsubstituted or unsubstituted furan, substituted or unsubstitutedthiophene, substituted or unsubstituted oxazole, substituted orunsubstituted isoxazole, substituted or unsubstituted indole,substituted or unsubstituted benzofuran, and substituted orunsubstituted benzo[b]thiophene, or

R³ and R⁴ together form a 6-membered heterocyclic ring selected fromsubstituted or unsubstituted piperidine, substituted or unsubstitutedpiperazine, substituted or unsubstituted thiane, substituted orunsubstituted morpholine, and substituted or unsubstitutedthiomorpholine, or

R³ and R⁴ together form a 6-membered heteroaryl ring selected fromsubstituted or unsubstituted pyridine, substituted or unsubstitutedpyridazine, substituted or unsubstituted pyrimidine, substituted orunsubstituted pyrazine, and substituted or unsubstituted morpholine, or

R³ and R⁴ together form a 6-membered aryl ring selected from substitutedor unsubstituted phenyl, substituted or unsubstituted biphenyl, andsubstituted or unsubstituted naphthyl.

In another embodiment, the disclosure provides a compound of Formula I,wherein:

R¹ is independently selected from hydroxyl;

R² is independently selected from (C₁-C₂₀)alkyl;

R³ is independently selected from hydroxyl; and

R⁴ is independently selected from hydrogen.

In another embodiment, the disclosure provides a method of identifying acompound that selectively kills polyploid cells, by:

converting a diploid cell line into a homogeneous population ofpolyploid cells;

screening one or more compounds against the homogeneous population ofpolyploid cells; and

identifying a compound that selectively kills the homogeneous populationof polyploid cells.

In another embodiment, the disclosure provides a method of identifying acompound that selectively kills polyploid cells, further including:exploiting Bcl-2 to protect the homogeneous population of polyploidcells from apoptosis and/or autophagic cell death when MYC-VX680synthetic lethality is elicited.

In another embodiment, the disclosure provides a method of identifying acompound that selectively kills polyploid cells, wherein the one or morecompounds has Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is independently selected from hydrogen, hydroxyl, amino, thiol,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl, alkyloxy,alkyloxyalkyl, alkylamine, dialkylamine, arylamine, heterocyclyl,alkylheterocyclyl, aryl, alkylaryl, and heteroaryl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy esteryl, and aryl;

R² is independently selected from alkyl, alkenyl, alkynyl, cycloalkyl,alkylcycloalkyl, alkoxy, and alkyloxyalkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy, esteryl, and aryl;

R³ and R⁴ are each independently selected from hydrogen, hydroxyl,amino, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl,alkyloxy, alkyloxyalkyl, alkylamine, dialkylamine, arylamine,heterocyclyl, heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, andheteroaryl, each optionally independently substituted with 1 to 6substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl,or aryl, or

R³ and R⁴ together form a 5 or 6-membered heteroaryl ring or a6-membered aryl ring, each optionally independently substituted with 1to 6 substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl,or aryl.

In another embodiment, the disclosure provides a method of identifying acompound that selectively kills polyploid cells, wherein:

R¹ is independently selected from hydrogen, hydroxyl, amino, thiol,(C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl,(C₅-C₂₀)cycloalkenyl, (C₈-C₂₀)cycloalkynyl,(C₁-C₂₀)alkyl(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkyl(C₅-C₂₀)cyclo-alkenyl,(C₁-C₂₀)alkyl(C₈-C₂₀)cycloalkynyl, (C₁-C₂₀)alkyloxy,(C₁-C₂₀)alkyloxy(C₁-C₂₀)alkyl, (C₁-C₂₀)alkylamine, (C₁-C₂₀)dialkylamine,arylamine, heterocyclyl, (C₁-C₂₀)alkylhetero-cyclyl, aryl,(C₁-C₂₀)alkylaryl, and heteroaryl, each optionally independentlysubstituted with 1 to 6 substituents selected from hydrogen, halogen,nitro, amino, cyano, isocyano, thiol, hydroxyl, (C₁-C₆)alkyl,(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, aryloxy, esteryl, and aryl;

R² is independently selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkyl(C₃-C₂₀)cycloalkyl,(C₁-C₂₀)alkoxy, and (C₁-C₂₀)alkyloxy(C₁-C₂₀)alkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,(C₁-C₂₀)alkyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, aryloxy, esteryl, andaryl;

R³ and R⁴ are each independently selected from hydrogen, hydroxyl,amino, thiol, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl,(C₃-C₂₀)cycloalkyl, (C₅-C₂₀)cycloalkenyl, (C₈-C₂₀)cycloalkynyl,(C₁-C₂₀)alkyl(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkyl(C₅-C₂₀)cycloalkenyl,(C₁-C₂₀)alkyl(C₈-C₂₀)cycloalkynyl, (C₁-C₂₀)alkyloxy,(C₁-C₂₀)alkyloxy(C₁-C₂₀)alkyl, (C₁-C₂₀)alkylamine, (C₁-C₂₀)dialkylamine,arylamine, heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, andheteroaryl, each optionally independently substituted with 1 to 6substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, (C₁-C₂₀)alkyl, (C₃-C₂₀)cycloalkyl,(C₁-C₂₀)alkoxy, aryloxy, esteryl, and aryl, or

R³ and R⁴ together form a 5 or 6-membered heterocyclic or heteroarylring or a 6-membered aryl ring, each optionally independentlysubstituted with 1 to 6 substituents selected from hydrogen, halogen,nitro, amino, cyano, isocyano, thiol, hydroxyl, (C₁-C₂₀)alkyl,(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, aryloxy, esteryl, and aryl.

In another embodiment, the disclosure provides a method of identifying acompound that selectively kills polyploid cells, wherein:

R³ and R⁴ together form a 5-membered heterocyclic ring selected fromsubstituted or unsubstituted pyrrolidine, substituted or unsubstitutedpyrazolidine, substituted or unsubstituted imidazolidine, substituted orunsubstituted tetrahydrofuran, and substituted or unsubstitutedtetrahydrothiophene, or

R³ and R⁴ together form a 5-membered heteroaryl ring selected fromsubstituted or unsubstituted furan, substituted or unsubstitutedthiophene, substituted or unsubstituted oxazole, substituted orunsubstituted isoxazole, substituted or unsubstituted indole,substituted or unsubstituted benzofuran, and substituted orunsubstituted benzo[b]thiophene, or

R³ and R⁴ together form a 6-membered heterocyclic ring selected fromsubstituted or unsubstituted piperidine, substituted or unsubstitutedpiperazine, substituted or unsubstituted thiane, substituted orunsubstituted morpholine, and substituted or unsubstitutedthiomorpholine, or

R³ and R⁴ together form a 6-membered heteroaryl ring selected fromsubstituted or unsubstituted pyridine, substituted or unsubstitutedpyridazine, substituted or unsubstituted pyrimidine, substituted orunsubstituted pyrazine, and substituted or unsubstituted morpholine, or

R³ and R⁴ together form a 6-membered aryl ring selected from substitutedor unsubstituted phenyl, substituted or unsubstituted biphenyl, andsubstituted or unsubstituted naphthyl.

In another embodiment, the disclosure provides a method of identifying acompound that selectively kills polyploid cells, wherein:

R¹ is independently selected from hydroxyl;

R² is independently selected from (C₁-C₂₀)alkyl;

R³ is independently selected from hydroxyl; and

R⁴ is independently selected from hydrogen.

In another embodiment, the disclosure provides a method of killingpolyploid tumor cells by administering to a patient in need thereof, acompound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is independently selected from hydrogen, hydroxyl, amino, thiol,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl, alkyloxy,alkyloxyalkyl, alkylamine, dialkylamine, arylamine, heterocyclyl,alkylheterocyclyl, aryl, alkylaryl, and heteroaryl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy esteryl, and aryl;

R² is independently selected from alkyl, alkenyl, alkynyl, cycloalkyl,alkylcycloalkyl, alkoxy, and alkyloxyalkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy, esteryl, and aryl;

R³ and R⁴ are each independently selected from hydrogen, hydroxyl,amino, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl,alkyloxy, alkyloxyalkyl, alkylamine, dialkylamine, arylamine,heterocyclyl, heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, andheteroaryl, each optionally independently substituted with 1 to 6substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl,or aryl, or

R³ and R⁴ together form a 5 or 6-membered heteroaryl ring or a6-membered aryl ring, each optionally independently substituted with 1to 6 substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl,or aryl.

In another embodiment, the disclosure provides a method of killingpolyploid tumor cells wherein:

R¹ is independently selected from hydrogen, hydroxyl, amino, thiol,(C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl,(C₅-C₂₀)cycloalkenyl, (C₈-C₂₀)cycloalkynyl,(C₁-C₂₀)alkyl(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkyl(C₅-C₂₀)cyclo-alkenyl,(C₁-C₂₀)alkyl(C₈-C₂₀)cycloalkynyl, (C₁-C₂₀)alkyloxy,(C₁-C₂₀)alkyloxy(C₁-C₂₀)alkyl, (C₁-C₂₀)alkylamine, (C₁-C₂₀)dialkylamine,arylamine, heterocyclyl, (C₁-C₂₀)alkylhetero-cyclyl, aryl,(C₁-C₂₀)alkylaryl, and heteroaryl, each optionally independentlysubstituted with 1 to 6 substituents selected from hydrogen, halogen,nitro, amino, cyano, isocyano, thiol, hydroxyl, (C₁-C₆)alkyl,(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, aryloxy, esteryl, and aryl;

R² is independently selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkyl(C₃-C₂₀)cycloalkyl,(C₁-C₂₀)alkoxy, and (C₁-C₂₀)alkyloxy(C₁-C₂₀)alkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,(C₁-C₂₀)alkyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, aryloxy, esteryl, andaryl;

R³ and R⁴ are each independently selected from hydrogen, hydroxyl,amino, thiol, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl,(C₃-C₂₀)cycloalkyl, (C₅-C₂₀)cycloalkenyl, (C₈-C₂₀)cycloalkynyl,(C₁-C₂₀)alkyl(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkyl(C₅-C₂₀)cycloalkenyl,(C₁-C₂₀)alkyl(C₈-C₂₀)cycloalkynyl, (C₁-C₂₀)alkyloxy,(C₁-C₂₀)alkyloxy(C₁-C₂₀)alkyl, (C₁-C₂₀)alkylamine, (C₁-C₂₀)dialkylamine,arylamine, heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, andheteroaryl, each optionally independently substituted with 1 to 6substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, (C₁-C₂₀)alkyl, (C₃-C₂₀)cycloalkyl,(C₁-C₂₀)alkoxy, aryloxy, esteryl, and aryl, or

R³ and R⁴ together form a 5 or 6-membered heterocyclic or heteroarylring or a 6-membered aryl ring, each optionally independentlysubstituted with 1 to 6 substituents selected from hydrogen, halogen,nitro, amino, cyano, isocyano, thiol, hydroxyl, (C₁-C₂₀)alkyl,(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, aryloxy, esteryl, and aryl.

In another embodiment, the disclosure provides a method of killingpolyploid tumor cells wherein:

R³ and R⁴ together form a 5-membered heterocyclic ring selected fromsubstituted or unsubstituted pyrrolidine, substituted or unsubstitutedpyrazolidine, substituted or unsubstituted imidazolidine, substituted orunsubstituted tetrahydrofuran, and substituted or unsubstitutedtetrahydrothiophene, or

R³ and R⁴ together form a 5-membered heteroaryl ring selected fromsubstituted or unsubstituted furan, substituted or unsubstitutedthiophene, substituted or unsubstituted oxazole, substituted orunsubstituted isoxazole, substituted or unsubstituted indole,substituted or unsubstituted benzofuran, and substituted orunsubstituted benzo[b]thiophene, or

R³ and R⁴ together form a 6-membered heterocyclic ring selected fromsubstituted or unsubstituted piperidine, substituted or unsubstitutedpiperazine, substituted or unsubstituted thiane, substituted orunsubstituted morpholine, and substituted or unsubstitutedthiomorpholine, or

R³ and R⁴ together form a 6-membered heteroaryl ring selected fromsubstituted or unsubstituted pyridine, substituted or unsubstitutedpyridazine, substituted or unsubstituted pyrimidine, substituted orunsubstituted pyrazine, and substituted or unsubstituted morpholine, or

R³ and R⁴ together form a 6-membered aryl ring selected from substitutedor unsubstituted phenyl, substituted or unsubstituted biphenyl, andsubstituted or unsubstituted naphthyl.

In another embodiment, the disclosure provides a method of killingpolyploid tumor cells, wherein:

R¹ is independently selected from hydroxyl;

R² is independently selected from (C₁-C₂₀)alkyl;

R³ is independently selected from hydroxyl; and

R⁴ is independently selected from hydrogen.

In another embodiment, the disclosure provides a combination oftherapeutic agents for use in treating a patient suffering from cancer,which includes:

a) at least one polyploidy inducing agent for use in inducingpolyploidization in one or more cancer cells in the patient; and

b) a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is independently selected from hydrogen, hydroxyl, amino, thiol,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl, alkyloxy,alkyloxyalkyl, alkylamine, dialkylamine, arylamine, heterocyclyl,alkylheterocyclyl, aryl, alkylaryl, and heteroaryl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy esteryl, and aryl;

R² is independently selected from alkyl, alkenyl, alkynyl, cycloalkyl,alkylcycloalkyl, alkoxy, and alkyloxyalkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy, esteryl, and aryl;

R³ and R⁴ are each independently selected from hydrogen, hydroxyl,amino, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl,alkyloxy, alkyloxyalkyl, alkylamine, dialkylamine, arylamine,heterocyclyl, heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, andheteroaryl, each optionally independently substituted with 1 to 6substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl,or aryl, or

R³ and R⁴ together form a 5 or 6-membered heteroaryl ring or a6-membered aryl ring, each optionally independently substituted with 1to 6 substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl,or aryl.

In another embodiment, the disclosure provides a combination oftherapeutic agents for use in treating a patient suffering from cancer,wherein the at least one polyploidy inducing agent is an Aurora Kinaseinhibitor that is specific to one type of aurora kinases or nonspecificto all three types of aurora kinases.

In another embodiment, the disclosure provides a method of treating apatient suffering from cancer, which includes:

a) administering to a patient suffering from cancer a therapeuticallyeffective amount of at least one polyploidy inducing agent, and

b) administering to the patient a therapeutically effective amount of acompound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is independently selected from hydrogen, hydroxyl, amino, thiol,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl, alkyloxy,alkyloxyalkyl, alkylamine, dialkylamine, arylamine, heterocyclyl,alkylheterocyclyl, aryl, alkylaryl, and heteroaryl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy esteryl, and aryl;

R¹ is independently selected from alkyl, alkenyl, alkynyl, cycloalkyl,alkylcycloalkyl, alkoxy, and alkyloxyalkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy, esteryl, and aryl;

R³ and R⁴ are each independently selected from hydrogen, hydroxyl,amino, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl,alkyloxy, alkyloxyalkyl, alkylamine, dialkylamine, arylamine,heterocyclyl, heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, andheteroaryl, each optionally independently substituted with 1 to 6substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl,or aryl, or

R³ and R⁴ together form a 5 or 6-membered heteroaryl ring or a6-membered aryl ring, each optionally independently substituted with 1to 6 substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl,or aryl.

In another embodiment, the disclosure provides a method of treating apatient suffering from cancer, wherein the at least one polyploidyinducing agent is an Aurora Kinase inhibitor.

Previously, a synthetic lethal interaction was found that allows theselective killing of cells that overexpress the Myc oncoprotein bypharmaceutical inhibitors of mitotic kinases, some of which are alreadyin clinical trials. This synthetic lethal interaction is attributable tothe inhibition of aurora-B kinase, with consequent disabling of thechromosomal passenger protein complex and ensuing DNA endoduplication;and executed by sequential apoptosis and autophagy. The results cast newlight on how such inhibitors kill cells, identify overexpression of Mycas a potential biomarker for tumor sensitivity to the inhibitors, andsuggest a therapeutic strategy that could mitigate current limitationson both the mitotic inhibitors as therapeutic agents and Myc as atherapeutic target.

It is conceivable that certain oncogenic alterations might modify thesynthetic lethal interaction, rendering it less effective in killing oftumor cells. Therefore, polyploid cells induced by the synthetic lethalinteraction might survive and accumulate, offering an invaluable modelsystem to screen for drugs that can selectively disrupt polyploid cellsas opposed diploid cells. This hypothesis was pursued by testingoncogenic elements that have been well known to promote survival inresponse to various stress stimuli.

Oncogenic alterations such as mutations/deletion of p53, overproductionof Bcl2 and IAPs, and constitutively active signaling from Ras and Akthave been associated with apoptosis-resistance of tumor cells tochemotherapeutic drugs. These alterations are known to attenuateMYC-dependent apoptosis under various conditions, act synergisticallywith Myc to elicit malignant transformation and are frequently found inhuman malignancies with deregulated expression of MYC. Whether theseoncogenic alterations have any impact on the MYC-dependent lethalityelicited by aurora kinase inhibitor VX-680 in RPE-MYC cells wasaddressed in a published study using a model cell line to demonstrateMYC-VX680 synthetic lethality.

The p53 function in RPE-Myc cells can be interfered with by either asmall molecule inhibitor of p53, PFT-a, (FIG. 1A), or a dominantnegative p53 mutant (FIG. 1B). Neither of these reagents protectedRPE-MYC cells from VX-680-induced cell death (FIG. 1A-B). In contrast,either of these reagents conferred more than 70% protection against thecytotoxicity elicited by hydroxyurea (FIG. 1A-B), confirming that thep53 function was compromised. The conclusion was that p53 was notrequired for the apoptosis elicited by VX-680 in cells overexpressingMYC.

Survival signals are often mediated by the Ras-PI3K-Akt pathway.Overexpression of an active Hu-Ras^(V12) elicited profound senescence inRPE-MYC cells, prevented generation of stable cell lines expressingHu-Ras^(V12), but were able to generate RPE-MYC cells expressing anactive allele of MyrAkt. RPE cells expressing both Myc and active Aktwere moderately more sensitive to the cytotoxicity of VX-680 than cellsexpressing Myc alone (FIG. 1C). Thus, active signaling from Aktenhanced, rather than blocked, the apoptosis elicited by VX-680. The BCLfamily antiapoptotic factor Bcl2 is a critical regulator ofmitochondrion-dependent apoptotic pathway by inhibiting cytochrome Crelease from mitochondria, whereas XIAP, the most characterized memberof the IAP family, has been known to sequester and inactivate activecaspases, proteases responsible for apoptosis. The overexpression ofBcl2 or Bcl-xL provided more than 40% protection against thecytotoxicity of VX-680 six days after administration of VX-680 (see,FIGS. 1C and 2) and that overexpression of XIAP has no effect on theMYC-dependent cytotoxicity (FIG. 1C). Since the apoptosis elicited byVX-680 in cells overexpressing MYC was p53-independent and could not beeffectively prevented by overexpression of antiapoptotic factors XIAPand an active Akt, the conclusion is that the MYC-dependent cytotoxicityelicited by VX-680 was likely mechanistically distinct from thepreviously reported MYC-dependent apoptosis in response to hypoxia,growth factor deprivation and genotoxic chemotherapeutic drugs.

Atg6/Beclin1 is part of a protein-kinase complex that participates information of autophagosomes. Atg6 is highly induced by Myc-VX680synthetic lethal interaction and is required for execution of thedelayed autophagic death. Since the proteins Bcl-2 and Bcl-X_(L)suppress autophagy through their interactions with Atg6, suppression ofMYC-VX680 synthetic lethality by Bcl-2 and Bcl-xL was tested to see ifit was mediated by its interaction with Beclin 1.

Wild-type Bcl-2 largely prevented delayed death of multinucleatedRPE-MYC cells (FIG. 1C and FIG. 2A-B). Furthermore, expression of aBcl-x_(L) mutant 1 (F131 V, D133A) that is defective in binding topro-apoptotic members of the Bcl-2 family, such as Bax, Bak and Bim,suppressed the death of multinucleated cells, indicating that theantiapoptotic function of Bcl-x_(L) is not required for rescuingmultinucleated cells from death (FIG. 2A-C).

By contrast, expression of a Bcl-2 mutant that fails to bind to Beclin1, Bcl-2 G145, was inert in this rescue experiment (FIG. 2A-B), despitesimilar levels of expression as wild-type Bcl-2 (FIG. 2C). Thus, adirect interaction with Atg6 was apparently required for Bcl-2 tosuppress nonapoptotic cell death of polyploid cells elicited by VX-680.In addition, a Bcl2 derivative that localizes to endoreticulum (Bcl2-ER)but not a version of Bcl2 that localizes to mitochondria (Bcl-2Mito)suppressed the delayed cell death, allowing accumulation of polyploidcells six days after initiation of VX680 treatment (FIG. 2B). Thus,suppression of Myc-VX680 synthetic lethality by Bcl-2 requires itsinteraction with Atg6 and localization to ER. This Bcl2Beclin1interaction likely occurs at ER.

Polyploidy is known to be associated with drug resistance. Butcurrently, there are no drugs in clinical trials that can kill polyploidcells. Since polyploid cells elicited by Myc-VX680 synthetic lethalitydo not die in the presence of Bcl-2, these cells provide a useful modelsystem to screen for small-molecule drugs that can selectively killpolyploid cells versus diploid cells. The VX680-resistant cell line wasnamed as RPEMYC/Bcl2. As a proof of concept experiment, polyploidRPEMYC/Bcl2 cells induced by VX680 were tested with a panel of 27 smallmolecule drugs including two Bcl-2 inhibitors. As expected all polyploidcells died within 48 hours of cells exposure to either of the two Bcl-2inhibitors (FIG. 3A).

By contrast, all diploid parental RPEMYC/Bcl2 cells survived thetreatment (FIG. 3B). For the remaining 25 drugs, none of them eliciteddeath of polyploid RPEMYC/Bcl2 cells (FIG. 3A). This model system wasexpected to allow the identification of other Bcl-2 inhibitors orinhibitors that block a function either upstream or downstream of Bcl-2.For this purpose, a library of 5000 extracts from 1000 different Chinesemedicinal herbs was established. One crude extract was identified thatdemonstrated potent killing of polyploid cells. Fractionation of thebioactive ingredient in the extracts led to identification ofhydroxybenzoquinone embelin (FIG. 4A). The selectively killing was alsoreproduced with commercially available embelin from Sigma (FIG. 4B).This killing by embelin was much more potent than Bcl2 inhibitors andeliminated most of polyploid cells within 24-hour of treatment (FIG.4B). In contrast, Bcl2 inhibitors failed to kill any polyploid cellswithin 24-hour of treatment (FIG. 4B), despite massive cell deathoccurred after 48 hours (FIG. 3A). A three-fold increase of Bcl2inhibitor dose also failed to elicit any death of polyploid cells within24 hours.

This contrast between embelin and Bcl2 inhibitors indicates that embelinmight elicit cell death in a mechanism different from that utilized byBcl2 inhibitors. The killing was not due to inhibition of the knownembelin target XIAP either, because depletion of XIAP with its esiRNAdid not affect viability of the polyploid cells. Currently, the exactmechanism underlying the killing of embelin is not known. Thepreliminary data point to the possibility that compromising oxidativephosphorylation of mitochondria by embelin is responsible for themassive disruption of polyploid tumor cells.

RPEMYC/BCL-2 is a human model cell line that has been transformed bythree defined oncogenic elements (Telomerase, Myc and Bcl-2). The cellline could form xenograft tumors in athymic mice. Likewise, polyploidRPEMYC/BCL2 cells induced by VX-680 in vitro can also readily formtumors upon implantation into athymic mice. The therapeutic efficacy ofembelin was compared against tumorigenesis of polyploid and diploidRPEMYC/BCL2 cells. The treatment with embelin was initiated immediatelyafter the implantation. This treatment dramatically delayed tumorformation of VX680 induced polyploid cells (FIG. 5A). However,tumorigenesis initiated by parental RPEMYC/BCL2 cells was largelyunaffected (FIG. 5A). A similar conclusion was reached with a breastcancer cell line MDA23MYC/Bcl2 that has been engineered to express bothMYC and Bcl2. Tumorigenesis of VX680-induced polyploid but not theirparental MDA231MYC/Bcl2 cells was delayed by the treatment with embelin(FIG. 5B). The therapeutic efficacy could be attributed to selectivelykilling of polyploid MDA231MYC/Bcl2 cells, rather than their parentalcells (FIG. 5C).

Lung cancer cell line MM-BRAF can form tumors with 100% penetrance atthe injection site of athymic mice and metastasize to various internalorgans such as liver, lung, kidney and spleen with 70-100% penetrance.Despite MM-BRAF having abundant expression of Myc, these tumor cellswere intrinsically resistant to MYC-VX680 synthetic lethality and failedto undergo cell death when exposed to VX-680 in vitro (FIG. 6A).Instead, all cells became multinucleated and polyploid after treatmentwith VX680 for 4 days (FIG. 6B). The mechanism underlying the VX680resistance has not yet been explored. These treatment-induced polyploidcells, like untreated parental cells, exhibited a very high frequency oftumor formation and metastasis (FIG. 6C-F). Implantation of just 3000polyploid cells were sufficient to form primary tumors at the injectionsite and initiate metastasis to various internal organs including theliver, lung and kidneys. Histologically, tumors formed by both polyploidcells and their parental cells were composed of small cells, rather thanlarge polyploid cells (FIG. 6C). The conclusion is that polyploidMM-BRAF cells have a very high frequency to revert back into diploidcells and re-enter cell division.

Treatment with embelin (unfilled columns), however, completely blockedmetastasis and drastically reduced tumor formation of polyploid MM-BRAFcells at the injection site (FIG. 7A). In contrast, tumorigenesis andmetastasis of parental MMBRAF cells was only modestly affected (FIG.7B). The contrast was mirrored by differential effect of embelin onpolyploid and diploid MM-BRAF in vitro. Embelin selectively killedpolyploid MM-BRAF cells but sparing their parental cells (FIG. 7C).

In another aspect, the present disclosure provides a pharmaceuticalcomposition, which includes a compound of Formula I in admixture with apharmaceutically acceptable excipient. One of skill in the art willrecognize that the pharmaceutical compositions include thepharmaceutically acceptable salts of the compound of Formula I asdescribed above.

In therapeutic and/or diagnostic applications, the compounds of theinvention can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington: The Science andPractice of Pharmacy (20^(th) ed.) Lippincott, Williams & Wilkins(2000).

The compounds according to the invention are effective over a widedosage range. For example, in the treatment of adult humans, dosagesfrom 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, andfrom 5 to 40 mg per day are examples of dosages that may be used. A mostpreferable dosage is 10 to 30 mg per day. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the subject to be treated, the body weight of the subjectto be treated, and the preference and experience of the attendingphysician.

It was previously found that synthetic lethal interaction between MYCand disabling the chromosomal passenger protein complex elicits DNAendoreduplication and apoptosis. Polyploid cells survived apoptosis buteventually killed by a form of nonapoptotic cell death termed asautophagic cell death. Now, it has been found that expression of eitherBcl-2 or Bcl-xL can rescue such polyploid cells from autophagic celldeath. Thus, the expression of other antiapoptotic members of the Bcl2family might serve the same purpose to confer resistance to MYC-VX680synthetic lethality and subsequently allow generation of polyploidcells. This discovery allowed creation of a cell-based screening assayto identify small molecules that can selectively kill polyploid cells,as opposed to diploid cells. Using this assay, a library of naturalproduct extracts was screened. Several extracts from the fruits ofEmbelia ribes Burm were discovered, and the bioactive ingredient fromthe extracts were further purified and finally identified thehydroxybenzoquinone embelin as the bioactive ingredient. With bothpurified and commercially available embelin, the therapeutic efficacy ofthe compound against multiple tumors such as breast cancer and lungcancer were demonstrated. The conclusion is that embelin may be usefulto be combined with therapeutics that can induce polyploidy to preventtumor relapse. Other derivatives of embelin might possess similar oreven better bioavailability and therapeutic efficacy than embelinitself.

EXAMPLES Materials and Methods Cell Lines and Culture Media

Various RPEMYC derivatives used in this study were generated by stablytransfecting human RPEMYC cells with a construct that expresses the geneof interest and selecting with 1 μg/ml of puromycin. Breast cancer cellline MD231MYC/Bcl2 was generated by engineering human MDA-MB-231 tostably express MYC and Bcl2. Lung cancer cell line MM-BRAF was createdfrom mouse lung cancer model initiated by BRAF^(V600E). All cell lineswere cultured in DMEM supplemented with 10% fetal bovine serum andantibiotics at 5% CO2 and 95% air in a humidified incubator.

Western Blot Analysis of Cell Extracts

Whole cell extracts were prepared by incubating cells for 15 min at 4°C. in a lysis buffer [50 mM Tris (pH 7.5), 200 mM NaCL, 0.1% SDS, 1%Triton X-100, 0.1 mM DTT, and 0.5 mM EGTA] supplemented with proteaseinhibitor mixture (BD Biosciences). The extracts were centrifuged at8,000× g for 10 min to clear insoluble material. The proteinconcentration in the supernatant was determined using the Bio-RadProtein Assay. Lysate containing 50-100 μg of proteins was resolved onNuPAGE (4-12%) Bis-Tris gels (Invitrogen) and transferred tonitrocellulose membranes (Bio-Rad). The membranes were blocked with 5%nonfat milk in PBS buffer for 1 h and then incubated overnight at 4° C.with primary antibodies diluted 1:1,000 in the blocking buffer. Rabbitpolyclonal antibodies were used for Bcl-2, Bcl-xL and PCNA. Horseradishperoxidaseconjugated anti-rabbit immunoglobulins were from Santa CruzBiotechnology. Western blots were developed with the SuperSignal WestFemto or Pico ECL detection kit (Thermo Scientific).

RNAi

XIAP esiRNAs were generated by digestion of double-stranded RNAcorresponding to the full-length coding sequence of the cognate geneswith Escherichia coli RNase III. The detailed protocol for preparationof esiRNA has been described elsewhere. esiRNAs smaller than 30 bp werepurified with DEAE columns and were transfected into cells withLipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions.

Small Molecules and Assays for Cell Death

Small molecules were purchased from commercial sources and used at theconcentrations indicated in figure legends and figures. Cell deathassays in response to drug treatment were performed 24 h after cellswere seeded in 24-well plates. Each experiment was done in triplicateand each experiment was repeated at least twice unless indicatedotherwise. Dead cells were identified by the trypan blue exclusionassay. All values are expressed as mean±1 SD of the mean.

Tumorigenicity Assays

Experimental mice were housed and treated according to the protocolapproved by the Institutional Animal Care and Use Committee of MBICR.Assays for tumorigenicity were performed by injecting 3000 to 5 millioncells in phosphate buffered saline (PBS) subcutaneously into the rightflanks of BALB/c athymic mice. A cohort of five to ten mice was used foreach group. In the treatment experiments, embelin 8 mg/kg was freshlydissolved in vehicle (10% DMSO, 100 mM Tris-HCl, pH 7.4) andadministered by intraperitoneal injection every 48 h. The control groupreceived injections of vehicle only. Tumor diameter at injection siteswas monitored at the indicated time points with a digital caliper andthe volume calculated using the formula: V (mm3)=A×B×B/2, where A and Brepresent the largest and smallest diameters of the tumor respectively.At the endpoint, mice were sacrificed to collect primary tumors andinternal organs for analysis of metastasis.

Tumor Tissue Processing and Histology

Tumors were dissected away from mice and then either frozen in liquidnitrogen or fixed in 4% paraformaldehyde at room temperature overnight.Processing of tissues and staining of tissue sections with hematoxylin &eosin were performed by using standard methods.

Statistical Analysis

Statistical analyses were performed with the GraphPad Prism software.Statistical significance of the differences was evaluated with Student'sunpaired two-tailed t test. P values less than 0.05 were consideredstatistically significant.

While the inventive features have been particularly shown and describedwith reference to preferred embodiments thereof, it will be understoodby those in the art that the foregoing and other changes may be madetherein without departing from the sprit and the scope of thedisclosure. Likewise, the various diagrams may depict an examplearchitectural or other configuration for the disclosure, which is doneto aid in understanding the features and functionality that can beincluded in the disclosure. The disclosure is not restricted to theillustrated example architectures or configurations but can beimplemented using a variety of alternative architectures andconfigurations. Additionally, although the disclosure is described abovein terms of various exemplary embodiments and implementations, it shouldbe understood that the various features and functionality described inone or more of the individual embodiments are not limited in theirapplicability to the particular embodiment with which they aredescribed. They instead can be applied alone or in some combination, toone or more of the other embodiments of the disclosure, whether or notsuch embodiments are described, and whether or not such features arepresented as being a part of a described embodiment. Thus, the breadthand scope of the present disclosure should not be limited by any of theabove-described exemplary embodiments.

1.-10. (canceled)
 11. A method of killing polyploid tumor cells,comprising: administering to a patient in need thereof, a compound ofFormula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ isindependently selected from hydrogen, hydroxyl, amino, thiol, alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl, alkyloxy,alkyloxyalkyl, alkylamine, dialkylamine, arylamine, heterocyclyl,alkylheterocyclyl, aryl, alkylaryl, and heteroaryl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy esteryl, and aryl; R² isindependently selected from alkyl, alkenyl, alkynyl, cycloalkyl,alkylcycloalkyl, alkoxy, and alkyloxyalkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy, esteryl, and aryl; R³ and R⁴ areeach independently selected from hydrogen, hydroxyl, amino, thiol,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl, alkyloxy,alkyloxyalkyl, alkylamine, dialkylamine, arylamine, heterocyclyl,heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, and heteroaryl, eachoptionally independently substituted with 1 to 6 substituents selectedfrom hydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy esteryl, or aryl, or R³ and R⁴together form a 5 or 6-membered heteroaryl ring or a 6-membered arylring, each optionally independently substituted with 1 to 6 substituentsselected from hydrogen, halogen, nitro, amino, cyano, isocyano, thiol,hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl, or aryl.
 12. Themethod of claim 11, wherein: R¹ is independently selected from hydrogen,hydroxyl, amino, thiol, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl,(C₃-C₂₀)cycloalkyl, (C₅-C₂₀)cycloalkenyl, (C₈-C₂₀)cycloalkynyl,(C₁-C₂₀)alkyl(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkyl(C₅-C₂₀)cyclo-alkenyl,(C₁-C₂₀)alkyl(C₈-C₂₀)cycloalkynyl, (C₁-C₂₀)alkyloxy,(C₁-C₂₀)alkyloxy(C₁-C₂₀)alkyl, (C₁-C₂₀)alkylamine, (C₁-C₂₀)dialkylamine,arylamine, heterocyclyl, (C₁-C₂₀)alkylhetero-cyclyl, aryl,(C₁-C₂₀)alkylaryl, and heteroaryl, each optionally independentlysubstituted with 1 to 6 substituents selected from hydrogen, halogen,nitro, amino, cyano, isocyano, thiol, hydroxyl, (C₁-C₆)alkyl,(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, aryloxy, esteryl, and aryl; R² isindependently selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkyl(C₃-C₂₀)cycloalkyl,(C₁-C₂₀)alkoxy, and (C₁-C₂₀)alkyloxy(C₁-C₂₀)alkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,(C₁-C₂₀)alkyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, aryloxy, esteryl, andaryl; R³ and R⁴ are each independently selected from hydrogen, hydroxyl,amino, thiol, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl,(C₃-C₂₀)cycloalkyl, (C₅-C₂₀)cycloalkenyl, (C₈-C₂₀)cycloalkynyl,(C₁-C₂₀)alkyl(C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkyl(C₅-C₂₀)cycloalkenyl,(C₁-C₂₀)alkyl(C₈-C₂₀)cycloalkynyl, (C₁-C₂₀)alkyloxy,(C₁-C₂₀)alkyloxy(C₁-C₂₀)alkyl, (C₁-C₂₀)alkylamine, (C₁-C₂₀)dialkylamine,arylamine, heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, andheteroaryl, each optionally independently substituted with 1 to 6substituents selected from hydrogen, halogen, nitro, amino, cyano,isocyano, thiol, hydroxyl, (C₁-C₂₀)alkyl, (C₃-C₂₀)cycloalkyl,(C₁-C₂₀)alkoxy, aryloxy, esteryl, and aryl, or R³ and R⁴ together form a5 or 6 membered heterocyclic or heteroaryl ring or a 6 membered arylring, each optionally independently substituted with 1 to 6 substituentsselected from hydrogen, halogen, nitro, amino, cyano, isocyano, thiol,hydroxyl, (C₁-C₂₀)alkyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, aryloxy,esteryl, and aryl.
 13. The method of claim 11, wherein: R³ and R⁴together form a 5-membered heterocyclic ring selected from substitutedor unsubstituted pyrrolidine, substituted or unsubstituted pyrazolidine,substituted or unsubstituted imidazolidine, substituted or unsubstitutedtetrahydrofuran, and substituted or unsubstituted tetrahydrothiophene,or R³ and R⁴ together form a 5-membered heteroaryl ring selected fromsubstituted or unsubstituted furan, substituted or unsubstitutedthiophene, substituted or unsubstituted oxazole, substituted orunsubstituted isoxazole, substituted or unsubstituted indole,substituted or unsubstituted benzofuran, and substituted orunsubstituted benzo[b]thiophene, or R³ and R⁴ together form a 6-memberedheterocyclic ring selected from substituted or unsubstituted piperidine,substituted or unsubstituted piperazine, substituted or unsubstitutedthiane, substituted or unsubstituted morpholine, and substituted orunsubstituted thiomorpholine, or R³ and R⁴ together form a 6-memberedheteroaryl ring selected from substituted or unsubstituted pyridine,substituted or unsubstituted pyridazine, substituted or unsubstitutedpyrimidine, substituted or unsubstituted pyrazine, and substituted orunsubstituted morpholine, or R³ and R⁴ together form a 6-membered arylring selected from substituted or unsubstituted phenyl, substituted orunsubstituted biphenyl, and substituted or unsubstituted naphthyl. 14.The method of claim 11, wherein: R¹ is independently selected fromhydroxyl; R² is independently selected from (C₁-C₂₀)alkyl; R³ isindependently selected from hydroxyl; and R⁴ is independently selectedfrom hydrogen.
 15. A combination of therapeutic agents for use intreating a patient suffering from cancer, comprising: a) at least onepolyploidy inducing agent for use in inducing polyploidization in one ormore cancer cells in the patient; and b) a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ isindependently selected from hydrogen, hydroxyl, amino, thiol, alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl, alkyloxy,alkyloxyalkyl, alkylamine, dialkylamine, arylamine, heterocyclyl,alkylheterocyclyl, aryl, alkylaryl, and heteroaryl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy esteryl, and aryl; R² isindependently selected from alkyl, alkenyl, alkynyl, cycloalkyl,alkylcycloalkyl, alkoxy, and alkyloxyalkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy, esteryl, and aryl; R³ and R⁴ areeach independently selected from hydrogen, hydroxyl, amino, thiol,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl, alkyloxy,alkyloxyalkyl, alkylamine, dialkylamine, arylamine, heterocyclyl,heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, and heteroaryl, eachoptionally independently substituted with 1 to 6 substituents selectedfrom hydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy esteryl, or aryl, or R³ and R⁴together form a 5 or 6-membered heteroaryl ring or a 6-membered arylring, each optionally independently substituted with 1 to 6 substituentsselected from hydrogen, halogen, nitro, amino, cyano, isocyano, thiol,hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl, or aryl.
 16. Thecombination of claim 15, wherein the at least one polyploidy inducingagent is an Aurora Kinase inhibitor that is specific to one type ofaurora kinases or nonspecific to all three types of aurora kinases. 17.A method of treating a patient suffering from cancer, comprising: a)administering to a patient suffering from cancer a therapeuticallyeffective amount of at least one polyploidy inducing agent, and b)administering to the patient a therapeutically effective amount of acompound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ isindependently selected from hydrogen, hydroxyl, amino, thiol, alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl, alkyloxy,alkyloxyalkyl, alkylamine, dialkylamine, arylamine, heterocyclyl,alkylheterocyclyl, aryl, alkylaryl, and heteroaryl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy esteryl, and aryl; R² isindependently selected from alkyl, alkenyl, alkynyl, cycloalkyl,alkylcycloalkyl, alkoxy, and alkyloxyalkyl, each optionallyindependently substituted with 1 to 6 substituents selected fromhydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy, esteryl, and aryl; R³ and R⁴ areeach independently selected from hydrogen, hydroxyl, amino, thiol,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,alkylcycloalkyl, alkylcycloalkenyl, alkylcycloalkynyl, alkyloxy,alkyloxyalkyl, alkylamine, dialkylamine, arylamine, heterocyclyl,heterocyclyl, alkylheterocyclyl, aryl, alkylaryl, and heteroaryl, eachoptionally independently substituted with 1 to 6 substituents selectedfrom hydrogen, halogen, nitro, amino, cyano, isocyano, thiol, hydroxyl,alkyl, cycloalkyl, alkoxy, aryloxy esteryl, or aryl, or R³ and R⁴together form a 5 or 6-membered heteroaryl ring or a 6-membered arylring, each optionally independently substituted with 1 to 6 substituentsselected from hydrogen, halogen, nitro, amino, cyano, isocyano, thiol,hydroxyl, alkyl, cycloalkyl, alkoxy, aryloxy esteryl, or aryl.
 18. Themethod of claim 17, wherein the at least one polyploidy inducing agentis an Aurora Kinase inhibitor.